Kristensen Jacob H, Karsdal Morten A, Sand Jannie Mb, Willumsen Nicholas, Diefenbach Claudia, Svensson Birte, Hägglund Per, Oersnes-Leeming Diana J
Nordic Bioscience A/S, Herlev, Denmark.
Department of Systems Biology, The Technical University of Denmark, Kgs. Lyngby, Denmark.
BMC Pulm Med. 2015 May 3;15:53. doi: 10.1186/s12890-015-0048-5.
During the pathological destruction of lung tissue, neutrophil elastase (NE) degrades elastin, one of the major constituents of lung parenchyma. However there are no non-invasive methods to quantify NE degradation of elastin. We selected specific elastin fragments generated by NE for antibody generation and developed an ELISA assay (EL-NE) for the quantification of NE-degraded elastin.
Monoclonal antibodies were developed against 10 NE-specific cleavage sites on elastin. One EL-NE assay was tested for analyte stability, linearity and intra- and inter-assay variation. The NE specificity was demonstrated using elastin cleaved in vitro with matrix metalloproteinases (MMPs), cathepsin G (CatG), NE and intact elastin. Clinical relevance was assessed by measuring levels of NE-generated elastin fragments in serum of patients diagnosed with idiopathic pulmonary fibrosis (IPF, n = 10) or lung cancer (n = 40).
Analyte recovery of EL-NE for human serum was between 85% and 104%, the analyte was stable for four freeze/thaw cycles and after 24 h storage at 4°C. EL-NE was specific for NE-degraded elastin. Levels of NE-generated elastin fragments for elastin incubated in the presence of NE were 900% to 4700% higher than those seen with CatG or MMP incubation or in intact elastin. Serum levels of NE-generated elastin fragments were significantly increased in patients with IPF (137%, p = 0.002) and in patients with lung cancer (510%, p < 0.001) compared with age- and sex-matched controls.
The EL-NE assay was specific for NE-degraded elastin. The EL-NE assay was able to specifically quantify NE-degraded elastin in serum. Serum levels of NE-degraded elastin might be used to detect excessive lung tissue degradation in lung cancer and IPF.
在肺组织的病理破坏过程中，中性粒细胞弹性蛋白酶（NE）可降解弹性蛋白，而弹性蛋白是肺实质的主要成分之一。然而，目前尚无用于定量NE对弹性蛋白降解作用的非侵入性方法。我们选择了由NE产生的特定弹性蛋白片段来制备抗体，并开发了一种酶联免疫吸附测定法（EL-NE）以定量NE降解的弹性蛋白。
针对弹性蛋白上10个NE特异性切割位点制备单克隆抗体。对一种EL-NE测定法进行分析物稳定性、线性以及批内和批间变异的检测。使用经基质金属蛋白酶（MMPs）、组织蛋白酶G（CatG）、NE体外切割的弹性蛋白以及完整弹性蛋白来证明NE的特异性。通过测量诊断为特发性肺纤维化（IPF，n = 10）或肺癌（n = 40）患者血清中NE产生的弹性蛋白片段水平来评估临床相关性。
EL-NE对人血清的分析物回收率在85%至104%之间，该分析物在四个冻融循环以及在4°C储存24小时后仍保持稳定。EL-NE对NE降解的弹性蛋白具有特异性。在NE存在下孵育的弹性蛋白中，NE产生的弹性蛋白片段水平比在CatG或MMP孵育或完整弹性蛋白中的水平高900%至4700%。与年龄和性别匹配的对照组相比，IPF患者（137%，p = 0.002）和肺癌患者（510%，p < 0.001）血清中NE产生的弹性蛋白片段水平显著升高。
EL-NE测定法对NE降解的弹性蛋白具有特异性。EL-NE测定法能够特异性地定量血清中NE降解的弹性蛋白。血清中NE降解的弹性蛋白水平可能用于检测肺癌和IPF中过度的肺组织降解。
